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Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids.

ABSTRACT: The hepatitis E virus (HEV) is an emerging pathogen showing a considerable increase in the number of reported cases in Europe mainly related to the ingestion of contaminated food. As with other relevant viral foodborne pathogens, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for HEV detection in clinical, food, and environmental samples, but these procedures cannot discriminate between inactivated and potentially infectious viruses. Thus, the aim of this study was to develop a viability PCR method to discriminate between native, heat-, and high-pressure processing (HPP)-treated HEV using the hepatitis A virus (HAV) as a cultivable surrogate. To this end, different concentrations of viability markers (PMAxx and platinum chloride, PtCl4) were screened firstly on purified viral RNA using different RT-qPCR assays. Reductions of HEV RNA signals of >17.5, >15.0, and >15.5 quantification cycles (Cq) were reported for PtCl4 and 1.6, 2.9, and 8.4 Cq for PMAxx, clearly indicating a better performance of PtCl4 than PMAxx irrespective of the RT-qPCR assay used. The most efficient viability pretreatment (500 ?M PtCl4 incubated at 5°C for 30 min) was then assessed on native, heat-, and HPP-treated HEV suspension. The optimized viability RT-qPCR discriminated successfully between native, heat-, and HPP-treated HEV, to different extents depending on the experimental conditions. In particular, approximately 2-log10 reduction was reported by PtCl4-RT-qPCR at both 72 and 95°C compared to the control. Additionally, both viability pretreatments were tested for HPP-treated HAV without success, while PtCl4-RT-qPCR completely eliminated (>5.6-log10 reduction) the RT-qPCR signals of HPP-treated HEV. Although this viability procedure may still overestimate infectivity, the PtCl4 pretreatment represents progress to better interpreting the quantification of intact HEV, and it could be included in molecular procedures used to quantify enteric viruses in food and environmental samples.

SUBMITTER: Randazzo W

PROVIDER: S-EPMC6119771 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids.

Randazzo Walter W Vasquez-García Andrea A Aznar Rosa R Sánchez Gloria G

Frontiers in microbiology 20180824

The hepatitis E virus (HEV) is an emerging pathogen showing a considerable increase in the number of reported cases in Europe mainly related to the ingestion of contaminated food. As with other relevant viral foodborne pathogens, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for HEV detection in clinical, food, and environmental samples, but these procedures cannot discriminate between inactivated and potentially infectious viruses. Thus, the aim of thi ...[more]

PMID: 30210465

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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Commonly used methods for both clinical diagnosis of SARS-CoV-2 infection and management of infected patients involve the detection of viral RNA, but the presence of infectious virus particles is unknown. Viability PCR (v-PCR) uses a photoreactive dye to bind non-infectious RNA, ideally resulting in the detection of RNA only from intact virions. This study aimed to develop and validate a rapid v-PCR assay for distinguishing intact and compromised SARS-CoV-2. Propidium monoazide (PMAxx) was used as a photoreactive dye. Mixtures with decreasing percentages of intact SARS-CoV-2 (from 100% to 0%) were prepared from SARS-CoV-2 virus stock and a clinical sample. Each sample was divided into a PMAxx-treated part and a non-PMAxx-treated part. Reverse transcription-PCR (RT-PCR) using an in-house developed SARS-CoV-2 viability assay was then applied to both sample sets. The difference in intact SARS-CoV-2 was determined by subtracting the cycle threshold (Ct) value of the PMAxx-treated sample from the non-PMAxx-treated sample. Mixtures with decreasing concentrations of intact SARS-CoV-2 showed increasingly lower delta Ct values as the percentage of intact SARS-CoV-2 decreased, as expected. This relationship was observed in both high and low viral load samples prepared from cultured SARS-CoV-2 virus stock, as well as for a clinical sample prepared directly from a SARS-CoV-2 positive nasopharyngeal swab. In this study, a rapid v-PCR assay has been validated that can distinguish intact from compromised SARS-CoV-2. The presence of intact virus particles, as determined by v-PCR, may indicate SARS-CoV-2 infectiousness.ImportanceThis study developed a novel method that can help determine whether someone who has been diagnosed with coronavirus disease 2019 (COVID-19) is still capable of spreading the virus to others. Current tests only detect the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, but cannot tell whether the particles are still intact and can thus infect cells. The researchers used a dye that selectively blocks the detection of damaged virions and free RNA. They showed that this viability PCR reliably distinguishes intact SARS-CoV-2 capable of infecting from damaged SARS-CoV-2 or free RNA in both cultured virus samples and a clinical sample. Being able to quickly assess contagiousness has important implications for contact tracing and safely ending isolation precautions. This viability PCR technique provides a simple way to obtain valuable information, beyond just positive or negative test results, about the actual risk someone poses of transmitting SARS-CoV-2 through the air or surfaces they come into contact with.

Project description:PurposeDecades of quality control efforts have raised the standards of immunohistochemistry (IHC), the principle method used for biomarker testing in breast cancer; however, computational pathology and reverse transcription quantitative PCR (RT-qPCR) may also hold promise for additional substantial improvements.MethodsHerein, we investigated discrepancies in the assessment of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and marker of proliferation Ki67 comparing routinely obtained IHC (and FISH) data (ORI) with the results of manual (REV) and semi-automated (DIA) re-evaluation of the original IHC slides and then with RNA expression data from the same tissue block using the MammaTyper® (MT) gene expression assay.ResultsCorrelation for ER and PR was high between ORI IHC and the other three study methods (REV, DIA and RT-qPCR). For HER2, 10 out of 96 discrepant cases can be detected between ORI and REV that involved at least one call in the equivocal category (except for one case). For Ki67, 22 (29.1%) cases were categorized differently by either REV alone (n = 17), DIA alone (n = 15) or both (n = 10) and 28 cases (29.2%) for RT-qPCR. Most of the discrepant Ki67 cases changed from low to high between the original and following assessment and belonged to the intermediate Ki67 expression range (between 9 and 30%).ConclusionsDetermination of the breast cancer biomarkers ER, PR, HER2 and Ki67 at the mRNA level shows high degree of correlation with IHC and compares well with correlations between original with subsequent independent manual or semi-automated IHC assessments. The use of methods with wider dynamic range and higher reproducibility such as RT-qPCR may offer more precise assessment of endocrine responsiveness, improve Ki67 standardization and help resolve HER2 cases that remain equivocal or ambiguous by IHC/FISH. In summary, our findings seem to configure RT-qPCR as a complementary method to be used in cases of either equivocal results or presenting, at the traditional determination assays, biomarkers expressions close to the cut-off values.

Dataset Information

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids.

Publications

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids.

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